Proper healing of corneal wounds is vital to maintaining a clear, healthy cornea and preserving vision. The long-term objective of this project is to understand the interaction of the corneal stroma and its keratocytes with corneal epithelium during wound repair. During the past two years we have observed proteins specific for each wound type synthesized after scrape, keratectomy, and burn wounds, and after epithelial sheets have been recombined onto wounded stromas. We observed approximately 14 proteins in wounded tissues not seen in controls. We selected three of these proteins to examine further for structure, function, and synthesis regulation. They are a 110K protein that appears only in epithelium migrating actively to cover wounds; a 70K glycoprotein that may play a role in adhesion during healing, since it exists on the epithelial-cell surface following wounding; and a 10-20K protein, which may have "hormone-like" qualities and is found in both the epithelium and stroma after the former has been recombined onto burned or keratectomized stroma. We will purify the three proteins, prepare monoclonal antibodies against the three proteins, immunolocalize the proteins in wounded corneas, and determine whether the mRNA levels of these three proteins increase after wounding, and whether 10-20K can affect corneal biosynthesis. The major purification tool will be HPLC. We will purify 110K using gel-filtration and ionic-exchange techniques, 10-20K in a single ionic exchange step, and 70K by concanavalin-A affinity chromatography and gel-filtration techniques. Monoclonal antibodies will be used to localize immunohistochemically the three proteins in tissue sections. Time-course studies of the appearance of the three proteins after wounding will determine when and where these proteins these proteins appear. To test for increased mRNA levels for 110K, 70K, and 10-20K after wounding, mRNA from control epithelium and stroma, and from epithelium and stroma harvested after scrape and keratectomy wounds will be isolated using guanidine isotheiocyanate extraction, ultracentrifugation through a CsC1 gradient, and selection by an oligo-(dT)-affinity column. mRNA levels for the proteins will be determined by cell-free translation of the mRNA from control and wounded tissues using a rabbit reticulocyte mix. The three proteins produced will be assayed using immunoprecipitation with three antibodies and SDS- PAGE. Clinically, our long-term goal is to study wound-healing mechanisms that can be used in therapies to enhance the wound- repair rate with minimal damage to the cornea.